(1) Field of the Invention
Tissues of patients are customarily examined for "markers" which may indicate a disease state. Marker evaluation can be an important part of a patient's initial diagnosis as well as provide a continued measurement of a patient's response to treatment and future prognosis. Conventional tissue markers include cell morphology and metabolism, the presence of certain enzymic activities or proteins or other molecules of biological import, the accumulation or disappearance of such molecules, etc. Recently, gene structure has provided a marker for certain genetic diseases (Geever et al., PNAS 78:5081-5085, 1981; Orkin et al. N. Eng.J. Med. 299:166-172, 1981, Chang and Kan PNAS 76:2886-2889, 1979; Philips et al., PNAS 77:2853-2856, 1980). However, until now it has not been possible to easily and economically measure primary gene activation, i.e., the accumulation of specific message RNA molecules (RNA transcription).
Having an appropriate "probe", usually a radiolabelled DNA molecule of known nucleotide sequence, it is possible to measure the quantity of specific message RNA species by a process called "molecular hybridization" (Gillespie, D., Methods Enzyme 12 B:641-668, 1968). Usually, the procedure involves first purifying message RNA from cells or tissues, a costly and laborious process. Molecular hybridizations utilizing purified RNA can be aided by immobilizing the RNA on a solid surface (Gilham, P. T., Biochemistry 7:2809-2813, 1968; Ponian, M. S., et al., Biochemistry 10:424-427, 1971; Wagner, A. F. et al., BBRC 45:184-189, 1971; Sheldon, R. et al., PNAS 69:417-421, 1972; Saxinger, W. C. et. al., PNAS 69:2975-2978, 1972; Noyes, B. E. and Stark, G. R., Cell 5:301-310, 1975; Alwine, J. C. et al., PNAS 74:5350-5354, 1977; Thomas, P. S., PNAS 77:5201-5205, 1980), still, RNA purification is required. Aternatively, cells can be deposited on microscope slides or a like surface and molecular hybridization can be performed upon the message RNA in the cells, a technique called " in situ molecular hybridization" (Pardue, M. L. and Gall, J. G., PNAS 64:600-604, 1969; Brahic, M. and Haase, A. T., PNAS 75:6125-6129, 1978; Angerer, L. M. and Angerer, R. C., Nuc. Ac. Res. 9:2819-2840, 1981). However, this process can be time-consuming and unreliable and is only useful when the assay can be performed upon single cells.
We have devised a process, "Direct RNA Immobilization", wherein whole cells are solubleized and passed through a filter. Most cellular constituents pass through the filter, but we have developed conditions where RNA becomes immobilized on the filter while other cell constituents which might confound molecular hybridization pass through or are inactiviated.
The process is not obvious, as evidenced by the lack of publications claiming to have developed the process. The process theoretically can be important in the diagnosis of any disease state which involves a change of RNA transcription in a tissue which can be biopsied; that is to say, virtually any disease. Practically speaking, the process is preferred over existing procedures when the existing procedures which could yield the same information are laborious, expensive or nonexistent.
We have discovered that RNA deposited on solid supports by Direct RNA Immobilization is in a suitable configuration to serve as a template for enzymatic synthesis of DNA, RNA and protein. This discovery has lead us to use Direct RNA Immobilization to develop new techniques in the field of Molecular Biology.
For example, the technique of cloning specific genes is limited by the process of screening recombinant clones for the gene of interest. When nucleic acid probes are available for the screening process, millions of clones can be screened. When no nucleic acid probes are available, under 100 clones can be screened and sometimes no screening is possible.
Using Direct RNA Immobilization we were able to develop a new cloning procedure where the screening of the clones is independent of the need for nucleic acid probes. Furthermore, the new process substantially improves the methodology for creating the initial clone bank.
(2) Description of the Prior Art
Gillespie and Spiegelman (J. Mol. Biol 12:829-842, 1965) developed a method for immobilizing purified DNA on nitrocellulose in a manner suitable for molecular hybridization. More recently Benton and Davis (Science 196:180-182, 1977), Grunstein and Hogness (PNAS 72:3961-3965, 1975), and Bresser and Gillespie (manuscript in preparation) learned how to deposit denatured DNA from dissolved viruses or cells onto nitrocellulose in a semiquantitative manner.
Several procedures have been developed over the years for the analogous immobilization of RNA (Gilham, P. T., Biochemistry 7:2809-2813, 1968; Ponian, M. S. et al., Biochemistry 10:424-427, 1971; Wagner, A. F. et al., BBRC 45:184-189, 1971; Sheldon, R. et al., PNAS 69:417-421, 1972; Saxinger, W. C. et al., PNAS 69:2975-2978, 1972; Noyes, B. E. and Stark, G. R., Cell 5:301-310, 1975; Alwine, J. C. et al., PNAS 74: 5350-5354, 1977; Thomas, P. S., PNAS 77:5201-5205, 1980). No process, save that described in this invention, demonstrates immobilization of RNA from dissolved cells. Furthermore, this invention utilizes a process and principles which are not indicated by the above referenced procedures for RNA immobilization.